Effect of Charge Reversal Mutations on the Ligand- and Membrane-binding Properties of Liver Fatty Acid-binding Protein
Liver fatty acid-binding protein (FABP) is able to bind to anionic phospholipid vesicles under conditions of low ionic strength. This binding results in the release of ligand, the fluorescent fatty acid analogue 11-dansylaminoundecanoic acid (DAUDA), with loss of fluorescence intensity (Davies, J. K...
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| Veröffentlicht in: | The Journal of biological chemistry 2002-12, Vol.277 (50), p.48395-48402 |
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| creator | Davies, Joanna K Hagan, Robert M Wilton, David C |
| description | Liver fatty acid-binding protein (FABP) is able to bind to anionic phospholipid vesicles under conditions of low ionic strength.
This binding results in the release of ligand, the fluorescent fatty acid analogue 11-dansylaminoundecanoic acid (DAUDA),
with loss of fluorescence intensity (Davies, J. K., Thumser, A. E. A., and Wilton, D. C. (1999) Biochemistry 38, 16932â16940). Using a strategy of charge reversal mutagenesis, the potential role of specific cationic residues in promoting
interfacial binding of FABP to anionic phospholipid vesicles has been investigated. Cationic residues chosen included those
within the α-helical region (Lys-20, Lys-31, and Lys-33) and those that make a significant contribution to the positive surface
potential of the protein (Lys-31, Lys-36, Lys-47, Lys-57, and Arg-126). Only three cationic residues make a significant contribution
to interfacial binding, and these residues (Lys-31, Lys-36, and Lys-57) are all located within the ligand portal region, where
the protein may be predicted to exhibit maximum disorder. The binding of tryptophan mutants, F3W, F18W, and C69W, to dioleoylphosphatidylglycerol
vesicles, containing 5 mol% of the fluorescent phospholipid dansyldihexadecanoylphosphatidylethanolamine, was monitored by
fluorescence resonance energy transfer (FRET). All three mutants showed enhanced dansyl fluorescence due to FRET on addition
of phospholipid to protein; however, this fluorescence was considerably greater with the F3W mutant, consistent with the N-terminal
region of the protein coming in close proximity to the phospholipid interface. These results were confirmed by succinimide
quenching studies. Overall, the results indicate that the portal region of liver FABP and specifically Lys-31, Lys-36, and
Lys-57 are involved in the interaction with the interface of anionic vesicles and that the N-terminal region of the protein
undergoes a conformational change, resulting in DAUDA release. |
| doi_str_mv | 10.1074/jbc.M208141200 |
| format | Article |
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This binding results in the release of ligand, the fluorescent fatty acid analogue 11-dansylaminoundecanoic acid (DAUDA),
with loss of fluorescence intensity (Davies, J. K., Thumser, A. E. A., and Wilton, D. C. (1999) Biochemistry 38, 16932â16940). Using a strategy of charge reversal mutagenesis, the potential role of specific cationic residues in promoting
interfacial binding of FABP to anionic phospholipid vesicles has been investigated. Cationic residues chosen included those
within the α-helical region (Lys-20, Lys-31, and Lys-33) and those that make a significant contribution to the positive surface
potential of the protein (Lys-31, Lys-36, Lys-47, Lys-57, and Arg-126). Only three cationic residues make a significant contribution
to interfacial binding, and these residues (Lys-31, Lys-36, and Lys-57) are all located within the ligand portal region, where
the protein may be predicted to exhibit maximum disorder. The binding of tryptophan mutants, F3W, F18W, and C69W, to dioleoylphosphatidylglycerol
vesicles, containing 5 mol% of the fluorescent phospholipid dansyldihexadecanoylphosphatidylethanolamine, was monitored by
fluorescence resonance energy transfer (FRET). All three mutants showed enhanced dansyl fluorescence due to FRET on addition
of phospholipid to protein; however, this fluorescence was considerably greater with the F3W mutant, consistent with the N-terminal
region of the protein coming in close proximity to the phospholipid interface. These results were confirmed by succinimide
quenching studies. Overall, the results indicate that the portal region of liver FABP and specifically Lys-31, Lys-36, and
Lys-57 are involved in the interaction with the interface of anionic vesicles and that the N-terminal region of the protein
undergoes a conformational change, resulting in DAUDA release.</description><identifier>ISSN: 0021-9258</identifier><identifier>EISSN: 1083-351X</identifier><identifier>DOI: 10.1074/jbc.M208141200</identifier><identifier>PMID: 12379651</identifier><language>eng</language><publisher>United States: American Society for Biochemistry and Molecular Biology</publisher><subject>Base Sequence ; Carrier Proteins - chemistry ; Carrier Proteins - genetics ; Carrier Proteins - metabolism ; Cell Membrane - metabolism ; DNA Primers ; Energy Transfer ; Fatty Acid-Binding Proteins ; Ligands ; Liver - metabolism ; Models, Chemical ; Mutagenesis, Site-Directed ; Neoplasm Proteins ; Phospholipids - metabolism ; Recombinant Proteins - chemistry ; Recombinant Proteins - genetics ; Recombinant Proteins - metabolism</subject><ispartof>The Journal of biological chemistry, 2002-12, Vol.277 (50), p.48395-48402</ispartof><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c360t-948779d07a3ad98f136181267439f469f405f3f588715e360e97e64437573ea23</citedby><cites>FETCH-LOGICAL-c360t-948779d07a3ad98f136181267439f469f405f3f588715e360e97e64437573ea23</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><link.rule.ids>314,776,780,27901,27902</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/12379651$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Davies, Joanna K</creatorcontrib><creatorcontrib>Hagan, Robert M</creatorcontrib><creatorcontrib>Wilton, David C</creatorcontrib><title>Effect of Charge Reversal Mutations on the Ligand- and Membrane-binding Properties of Liver Fatty Acid-binding Protein</title><title>The Journal of biological chemistry</title><addtitle>J Biol Chem</addtitle><description>Liver fatty acid-binding protein (FABP) is able to bind to anionic phospholipid vesicles under conditions of low ionic strength.
This binding results in the release of ligand, the fluorescent fatty acid analogue 11-dansylaminoundecanoic acid (DAUDA),
with loss of fluorescence intensity (Davies, J. K., Thumser, A. E. A., and Wilton, D. C. (1999) Biochemistry 38, 16932â16940). Using a strategy of charge reversal mutagenesis, the potential role of specific cationic residues in promoting
interfacial binding of FABP to anionic phospholipid vesicles has been investigated. Cationic residues chosen included those
within the α-helical region (Lys-20, Lys-31, and Lys-33) and those that make a significant contribution to the positive surface
potential of the protein (Lys-31, Lys-36, Lys-47, Lys-57, and Arg-126). Only three cationic residues make a significant contribution
to interfacial binding, and these residues (Lys-31, Lys-36, and Lys-57) are all located within the ligand portal region, where
the protein may be predicted to exhibit maximum disorder. The binding of tryptophan mutants, F3W, F18W, and C69W, to dioleoylphosphatidylglycerol
vesicles, containing 5 mol% of the fluorescent phospholipid dansyldihexadecanoylphosphatidylethanolamine, was monitored by
fluorescence resonance energy transfer (FRET). All three mutants showed enhanced dansyl fluorescence due to FRET on addition
of phospholipid to protein; however, this fluorescence was considerably greater with the F3W mutant, consistent with the N-terminal
region of the protein coming in close proximity to the phospholipid interface. These results were confirmed by succinimide
quenching studies. Overall, the results indicate that the portal region of liver FABP and specifically Lys-31, Lys-36, and
Lys-57 are involved in the interaction with the interface of anionic vesicles and that the N-terminal region of the protein
undergoes a conformational change, resulting in DAUDA release.</description><subject>Base Sequence</subject><subject>Carrier Proteins - chemistry</subject><subject>Carrier Proteins - genetics</subject><subject>Carrier Proteins - metabolism</subject><subject>Cell Membrane - metabolism</subject><subject>DNA Primers</subject><subject>Energy Transfer</subject><subject>Fatty Acid-Binding Proteins</subject><subject>Ligands</subject><subject>Liver - metabolism</subject><subject>Models, Chemical</subject><subject>Mutagenesis, Site-Directed</subject><subject>Neoplasm Proteins</subject><subject>Phospholipids - metabolism</subject><subject>Recombinant Proteins - chemistry</subject><subject>Recombinant Proteins - genetics</subject><subject>Recombinant Proteins - metabolism</subject><issn>0021-9258</issn><issn>1083-351X</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2002</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNpNkMFLwzAUh4Mobk6vHiUHr51JkzTJcYxNhQ1FFLyFtH1pM7Z2pNlk_70dG-iD997l-_0OH0L3lIwpkfxplRfjZUoU5TQl5AINKVEsYYJ-X6IhISlNdCrUAN103Yr0wzW9RgOaMqkzQYdoP3MOiohbh6e1DRXgD9hD6OwaL3fRRt82HW4bHGvAC1_Zpkxwf_ASNnmwDSS5b0rfVPg9tFsI0UN37Fr4vgTPbYwHPCl8-R-L4JtbdOXsuoO78x-hr_nsc_qSLN6eX6eTRVKwjMREcyWlLom0zJZaOcoyqmiaSc6041m_RDjmhFKSCugjoCVknDMpJAObshEan3qL0HZdAGe2wW9sOBhKzFGg6QWaP4F94OEU2O7yDZR_-NlYDzyegNpX9Y8PYHLfFjVsTCqlEcRwxbRgv0Qedrw</recordid><startdate>20021213</startdate><enddate>20021213</enddate><creator>Davies, Joanna K</creator><creator>Hagan, Robert M</creator><creator>Wilton, David C</creator><general>American Society for Biochemistry and Molecular Biology</general><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope></search><sort><creationdate>20021213</creationdate><title>Effect of Charge Reversal Mutations on the Ligand- and Membrane-binding Properties of Liver Fatty Acid-binding Protein</title><author>Davies, Joanna K ; Hagan, Robert M ; Wilton, David C</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c360t-948779d07a3ad98f136181267439f469f405f3f588715e360e97e64437573ea23</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2002</creationdate><topic>Base Sequence</topic><topic>Carrier Proteins - chemistry</topic><topic>Carrier Proteins - genetics</topic><topic>Carrier Proteins - metabolism</topic><topic>Cell Membrane - metabolism</topic><topic>DNA Primers</topic><topic>Energy Transfer</topic><topic>Fatty Acid-Binding Proteins</topic><topic>Ligands</topic><topic>Liver - metabolism</topic><topic>Models, Chemical</topic><topic>Mutagenesis, Site-Directed</topic><topic>Neoplasm Proteins</topic><topic>Phospholipids - metabolism</topic><topic>Recombinant Proteins - chemistry</topic><topic>Recombinant Proteins - genetics</topic><topic>Recombinant Proteins - metabolism</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Davies, Joanna K</creatorcontrib><creatorcontrib>Hagan, Robert M</creatorcontrib><creatorcontrib>Wilton, David C</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><jtitle>The Journal of biological chemistry</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Davies, Joanna K</au><au>Hagan, Robert M</au><au>Wilton, David C</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Effect of Charge Reversal Mutations on the Ligand- and Membrane-binding Properties of Liver Fatty Acid-binding Protein</atitle><jtitle>The Journal of biological chemistry</jtitle><addtitle>J Biol Chem</addtitle><date>2002-12-13</date><risdate>2002</risdate><volume>277</volume><issue>50</issue><spage>48395</spage><epage>48402</epage><pages>48395-48402</pages><issn>0021-9258</issn><eissn>1083-351X</eissn><abstract>Liver fatty acid-binding protein (FABP) is able to bind to anionic phospholipid vesicles under conditions of low ionic strength.
This binding results in the release of ligand, the fluorescent fatty acid analogue 11-dansylaminoundecanoic acid (DAUDA),
with loss of fluorescence intensity (Davies, J. K., Thumser, A. E. A., and Wilton, D. C. (1999) Biochemistry 38, 16932â16940). Using a strategy of charge reversal mutagenesis, the potential role of specific cationic residues in promoting
interfacial binding of FABP to anionic phospholipid vesicles has been investigated. Cationic residues chosen included those
within the α-helical region (Lys-20, Lys-31, and Lys-33) and those that make a significant contribution to the positive surface
potential of the protein (Lys-31, Lys-36, Lys-47, Lys-57, and Arg-126). Only three cationic residues make a significant contribution
to interfacial binding, and these residues (Lys-31, Lys-36, and Lys-57) are all located within the ligand portal region, where
the protein may be predicted to exhibit maximum disorder. The binding of tryptophan mutants, F3W, F18W, and C69W, to dioleoylphosphatidylglycerol
vesicles, containing 5 mol% of the fluorescent phospholipid dansyldihexadecanoylphosphatidylethanolamine, was monitored by
fluorescence resonance energy transfer (FRET). All three mutants showed enhanced dansyl fluorescence due to FRET on addition
of phospholipid to protein; however, this fluorescence was considerably greater with the F3W mutant, consistent with the N-terminal
region of the protein coming in close proximity to the phospholipid interface. These results were confirmed by succinimide
quenching studies. Overall, the results indicate that the portal region of liver FABP and specifically Lys-31, Lys-36, and
Lys-57 are involved in the interaction with the interface of anionic vesicles and that the N-terminal region of the protein
undergoes a conformational change, resulting in DAUDA release.</abstract><cop>United States</cop><pub>American Society for Biochemistry and Molecular Biology</pub><pmid>12379651</pmid><doi>10.1074/jbc.M208141200</doi><tpages>8</tpages><oa>free_for_read</oa></addata></record> |
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| source | MEDLINE; Elektronische Zeitschriftenbibliothek - Frei zugängliche E-Journals; Alma/SFX Local Collection |
| subjects | Base Sequence Carrier Proteins - chemistry Carrier Proteins - genetics Carrier Proteins - metabolism Cell Membrane - metabolism DNA Primers Energy Transfer Fatty Acid-Binding Proteins Ligands Liver - metabolism Models, Chemical Mutagenesis, Site-Directed Neoplasm Proteins Phospholipids - metabolism Recombinant Proteins - chemistry Recombinant Proteins - genetics Recombinant Proteins - metabolism |
| title | Effect of Charge Reversal Mutations on the Ligand- and Membrane-binding Properties of Liver Fatty Acid-binding Protein |
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